Solid-phase detection of terminal monosaccharides cleaved from glycosylated substrates

ABSTRACT

The present invention relates to a novel method for analysing carbohydrates. The invention is in particular useful in detecting a terminal monosaccharide which may be released from a glycosylated substrate for example using an exoglucosidase. After relase from the glycosylated substrate the terminal monosaccharide may be captured on a solid support, incubated with a boronate detection agent and detected by aid of the boronate detection agent. The methods of the invention are useful for a variety of purposes including sequencing of carbohydrates, wherein exoglycosidases with predetermined specificity are employed for the release.

FIELD OF INVENTION

The present invention relates to the field of carbohydrate analysis. Inparticular, the invention relates to new methods for identifying theterminal sugar-residues of carbohydrate chains, and for sequencingcarbohydrates, such as carbohydrates found in biological samples. Thus,in one aspect the invention relates to the field of carbohydratedetection and structural characterisation.

BACKGROUND OF THE INVENTION

Carbohydrates exist in many forms in nature. In animals including man,examples include free reducing sugars in solution (such as themonosaccharide glucose in serum), free oligosaccharides in solution(such as the disaccharide lactose in milk), they can be attached topeptides or proteins through covalent linkages to a variety of aminoacids (such as asparagine, serine, threonine and others), covalentlyattached to lipids such as ceramide (as in gangliosides) or attached tomembrane anchors via phosphatidylinositols. Sugars are also foundattached to many small molecules including some involved in metabolism,such as glucuronides. In the above examples, the length of the sugarchains can vary from one to over 100 sugar residues.

In lower organisms, including bacteria and plants, an even wider arrayof structures exists. The surface of bacterial cells can be covered bysugar polymers that are thousands of residues long, which can act asantigens in the detection of bacteria and as vaccines. Sugars are anintegral part of bacterial cell walls. The sugars can themselves beantibiotics (such as the aminoglycoside antibiotics, for examplestreptomycin), or can be found as essential components of antibiotics(such as erythromycin and vancomycin), as enzyme inhibitors (as inAcarbose) or as anti-cancer agents (such as for example calicheamycin).

One area of particular interest is the structure of the carbohydratechains (glycans) found attached to glycoproteins and glycolipids. Theglycosylation pattern of glycoproteins has been shown to be importantfor their biological functions, including their bioavailablity, theirtargeting, and have even been directly correlated with the metastaticpotential of tumor cells. The glycosylation pattern of human serumtransferrin, for example, is being used as a diagnostic test for aseries of genetic diseases termed Carbohydrate-Deficient GlycosylationSyndromes. Specific glycolipid sequences have been shown to be involvedin neuronal development and cell surface signalling, in diabetes, andare accumulated in certain specific metabolic diseases such asTay-Sachs, for which they are diagnostic.

The linkages between the sugar residues in the oligosaccharides andpolysaccharides described above can have either the alpha or betaconfigurations, and the glycans can be multiply branched. The diversityof structures possible for glycan chains is therefore enormous and theirstructural characterization is therefore inherently complex. There istherefore a strong interest in methods for the detection, structuralcharacterization, identification, quantitation, and chemical/enzymaticmanipulation of carbohydrate and glycan structures, in research, indiagnostics, in monitoring the glycosylation of recombinantglycoproteins and in the development of new pharmaceutical agents. Inthis last context, the degree of terminal galactosylation and sialyationof the glycan chains of recombinant glycoprotein drugs such aserythreopoetin is critically important for its effectiveness.

Several methods are in current use for the analysis for carbohydratestructures, and these have recently been reviewed. Underivatizedoligosaccharides and glycolipids can be analyzed by NMR-spectroscopy, bymass-spectrometry, and by chromatography. For the much largerglycoproteins, mass spectrometry provides more limited information butanalysis of their proteolytic digests, i.e. glycopeptides, has beenextensively used. Indirect structural information about underivatizedoligosaccharides can also be deduced from their abilities to interactwith carbohydrate-binding proteins such as lectins, antibodies orenzymes.

Carbohydrates themselves have no characteristic chromophores, onlyN-acetyl groups, so monitoring their separation by optical orspectroscopic detection is not commonly used. Pulsed amperometricdetection of the polyols has however been an important technique fordetection in chromatography. This technique has also been applied to thedetection and identification of monosaccharides in solution.

The most widely used method for high-sensitivity detection ofcarbohydrates has been the labeling of the reducing ends (lactols,tautomers of hydroxyaldehydes and hydroxyketones) with eitherradioactive or fluorescent TAGs. Both chemical and enzymatic methodshave been described that cleave carbohydrates from glycoproteins andglycolipids, permitting the generation of the required reducing sugarsfrom glycoproteins (including monosaccharides released byexo-glycosidases or acid-hydrolysis), glycolipds and otherglycoconjugates. Most commonly, such reducing sugars are reacted withamino-containing derivatives of fluorescent molecules under conditionsof reductive amination: i.e., where the initially formed imines (C═N)are reduced to amines (CH—NH) to produce a stable linkage. In mostcases, the labeling reactions have been performed in solution using alarge excess of labeling agent. This requires separation of the excesslabeling agent and its by-products prior to or during analysis. OtherTAGs of utility in mass-spectrometry have been added in the same manner,by either amination or reductive amination, the detection then beingperformed by the mass-spectrometer.

Once the label has been added to permit specific detection, thecarbohydrates (including monosaccharides) described above cansubsequently be subjected to separation and detection/quantification. Ifspecific glycosidases act on the tagged carbohydrates, they can cleaveone or more sugar residues resulting in a change in chromatographic orelectrophoretic mobility, as detected by, for example, a fluorescencedetector in HPLC, CE or by a change in their mobility in SDS-PAGE, or achange in their mass as detected by a change in m/z value in amass-spectrometer. Arrays of enzymes have been used to provide a higherthroughput analysis.

Below a short overview of prior art is given:

Gao et al. 2003 reviews suitable techniques for derivatisation ofcarbohydrates in solution. In solution carbohydrates may be derivatisedby reductive amination. In general, —NH₂ groups of amines may react withaldehyde or ketone group of reducing sugars, thereby producing compoundsof —C═N structure. Such compounds may further be reduced for example byNaCNBH₃. Gao et al., 2003 does not disclose the capture or detection ofterminal monosaccharides released from glycosylated substrates.

U.S. Pat. No. 5,100,778 describes a method for oligosaccharidesequencing comprising placing an identifying label on the reducingterminal residue of an oligosaccharide, dividing into a plurality ofseparate portions, treating each portion with for example specificglycosidases, pooling product and analysing the pools obtained. Thedocument does not describe immobilised terminal monosaccharides.

U.S. Pat. No. 4,419,444 describes methods for chemically binding organiccompounds containing carbohydrate residues to a support bearing reactive—NH₂ groups. The methods involve either the periodate oxidation ofcarbohydrate diols to produce reactive aldehydes by cleaving of C—Cbonds in the carbohydrate or oxidation of —CH₂OH groups to —CHO groupsenzymatically. Both oxidations will result in alteration of thestructure of the carbohydrate. The reactive aldehydes can be immobilisedby reaction with the —NH₂ groups. After immobilisation of thecarbohydrate containing compound a reduction step (for example usingNaBH₄) may be performed to increase stability. The document does notdescribe the immobilization of a single monosaccharide after cleavagefrom a carbohydrate-containing compound. Furthermore, the chemicalnature of the carbohydrate has been altered and this alteration mayimpair further modulations, such as specific enzymatic cleavage byglycosidases. The document also does not describe the addition of anychemical reagents to the immobilised carbohydrates that result in theaddition of molecular structures to it.

WO92/719974 describes a method of sequencing oligosaccharides. Themethod involves immobilising oligosaccharides on a solid support andsubsequent treatment with a variety of glycosidases. Prior toimmoblisation, the oligosaccharide may be linked to a conjugate. Thedocument does not describe modulation of immobilised terminalmonosaccharides, nor indeed treatment with the tagged compounds of thepresent invention.

Lohse et al. (“Solid-Phase Oligosaccharide Tagging (SPOT): Validation onGlycolipid-Derived Structures”, Angew. Chem. Int. Ed. 2006, 45,4167-4172) disclose Solid-Phase Oligosaccharide Tagging onglycolipid-derived structures, however this article does not disclosecleavage of terminal monosaccharides from glycosylated substrates beforeanalysis of the immobilised monosaccharides, nor indeed the use of thespecific boronate compounds disclosed herein.

Various boronate compounds have been previously used for labelling anddetecting carbohydrates in solution, such as disclosed in e.g. thefollowing references:

-   -   “Boronic Acids: Preparation, Applications in Organic Synthesis        and Medicine”, ed. Dennis G. Hall, pub. Wiley-VCH, in particular        in chapters 12 and 13 (“Boronic Acid-based receptors and sensors        for saccharides” and “Biological and medicinal applications of        boronic acids”).    -   Yan et al., “Boronolectins and Fluorescent Boronolectins: An        Examination of the Detailed Chemistry Issues Important for the        Design”, Medicinal Reseach Reviews, Vol. 25, No. 5, 490-520,        2005    -   Mulla et al., “3-Methoxycarbonyl-5-nitrophenyl boronic acid:        high affinity diol recognition at neutral pH”, Bioorganic &        Medicinal Chemistry Letter 14 (2004) 25-27

-   Dowlut et al., “An Improved Class of Sugar-Binding Boronic Acids,    Soluble and Capable of Complexing Glycosides in Neutral Water”, J.    Am. Chem. Soc. 2006, 128, 4226-7    -   Hoeg-Jensen., “Preparation and Screening of Diboronate Arrays        for Identification of Carbohydrate Binders”, QSAR Comb., Sci.        2004, 23    -   Boduroglu et al., “A colorimetric titration method for        quantification of millimolar glucose in a pH 7.4 aqueous        phosphate buffer”, Bioorganic & Medicinal Chemistry Letter        15 (2005) 3974-3977    -   Davis et al., “Simple and Rapid Visual Sensing of Saccharides”,        Organic Letter, 1999 Vol. 1, No. 2, 331-334    -   He et al., “Chromophore Formation in Resorcinarene Solutions and        the Visual detection of Mono- and Oligosaccharides”, J. Am.        Chem. Soc. 2003, 124, 5000-5009    -   Gray et al., “Specific sensing between inositol epimers by a        bis(boronate)”, Bioorganic & Medicinal Chemistry Letters        15 (2005) 5416-5418)

However, none of the above-mentioned compounds have been used to labeland detect carbohydrates immobilised to a solid support, nor indeedterminal monosaccharides attached to a solid support.

The above sections describe the biological importance and complexity ofglycans, and summarizes some benefits of attaching TAGs such asboronates to sugars, including monosaccharides, although not toimmobilised terminal monosaccharides after cleavage from acarbohydrate-containing molecule. To date, such TAG attachment has onlybeen performed in solution using large excesses of tagging agent (andoften additional chemical agents such as reducing agents), and thusrequire time consuming and frequently difficult separation techniques tobe applied before either detection or further manipulation. There istherefore a great need for simple methods that can allow easiercarbohydrate sequencing through identification of a terminalmonosaccharide, without the need for complex methods for separatingreaction starting materials, reagents, by-products and sought afterproducts. We describe herein such simple methods.

SUMMARY OF THE INVENTION

In solution monosaccharides are found primarily in cyclic forms. Thus,in solution aidohexoses are for example primarily present as pyranoseswith a smaller fraction being present as furanoses. Only a very minorfraction is present as open chain aldehyde or hydrate (see for exampleZhu et al., 2001, J. org. Chem, 66:6244-6251). Interestingly, Dowlut etal., 2006, J. Am. Chem. Soc, 128: 4226-4227 states that “no boronic acidunit has yet been demonstrated to bind to nonreducing sugars andglycosides” (p. 4226, 1^(st) col. I. 8-10). The document furthermorediscloses a specific orthosubstituted aryl boronic acid which only bindsto glycosides with an extremely low affinity (K_(a) in the range of22-34 M⁻¹). Accordingly, in solution in the absence of a reducing agentonly a very minor fraction of monosaccharides are capable of interactingwith boronates.

As is apparent from the above, the particular conformational structureof a sugar is very important for the ability to interact with boronicacid or boranates. The prior art does not describe or hint at whetherimmobilised sugars may adopt a conformation allowing binding of boronicacid or boronates. Interestingly, the present invention discloses thatboronates may be useful in the detection of immobilised terminalmonosaccharides. In fact the present invention discloses thatimmobilised terminal monosaccharides may even be visually detected usingtagged boronates.

Thus, the present invention provides a method for analysis of a terminalmonosaccharide on a glycosylated substrate, said method comprising thesteps of:—

-   (i) detaching said monosaccharide from said glycosylated substrate,    preferably using an exoglycosidase;-   (ii) allowing said detached monosaccharide to covalently bind to a    capture group on a solid support;-   (iii) incubating said covalently bound monosaccharide with a    detection agent with formula X:

TAG-R-Boronate

wherein TAG=a tag moiety capable of being detected

-   -   R=organic moiety    -   Boronate=a boronic acid moiety or ester thereof,        said boronate being attached to a carbon atom comprised in said        R group;

-   (iv) allowing the detection agent to bind the monosaccharide

-   (v) detecting detection agent having bound to the monosaccharide.

Preferably, said detection agent has formula selected from:TAG-R—B(OH)₂, TAG-R—B(OH)(OR′) or TAG-R—B(OR′)(OR″)

wherein R′and R″ may be either aliphatic or aromatic and are optionallycovalently attached to R.

The steps comprised in said method can be repeated at least once (asdescribed further herein below), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more times, in order to allow efficient sequencing ofmonosaccharide(s) comprised in the glycosylated substrate. The methodcan also be carried out on different carbohydrate chains on the sameglycosylated substrate, and/or repeated more than once.

The methods of the present invention may be applied to the detection,identification and quantitation of terminal glycosylation pattern ofrecombinant glycoproteins such as biopharmaceuticals, for example foridentifying and quantifying terminal glycose residues that are presenton glycosylated substrates such as glycoproteins, glycopeptides,glycolipids, polysaccharides and oligosaccharides. In particular, themethod is shown to distinguish between terminally sialyatedglycoproteins and terminally galactosylated glycoproteins.

Thus, the methods of the present invention may in one embodiment be usedto identify particular glycosylation patterns associated with certainpathological conditions. Therefore, in a further aspect of the presentinvention is disclosed a method of diagnosis of a disease associatedwith abnormal glycoprotein glycosylation, comprising subjecting a sampleof glycoproteins obtained from a patient to a method as disclosedherein.

The methods of the present invention may also be advantageously used toassay for bacterial contamination of products, thus in another aspect ofthe present invention is provided a method for monitoring for bacterialcontamination of products, such as pharmaceutical products, comprisingsujecting a sample of said product to one of the methods disclosedherein.

Further disclosed herein are detection agents suitable for use in themethods of the present invention, as well as covalent adducts formedbetween a monosaccharide and said detection agent(s). Furthermore,fluorescent compounds suitable for use in the detection agents accordingto the present invention are disclosed.

In another aspect of the present invention is disclosed a kit of partssuitable for using in the methods of the present invention, said kit ofparts comprising at least one solid support, at least one capture group,and one or more of the detection agents disclosed herein.

DESCRIPTION OF DRAWINGS

FIG. 1. Release of a terminal monosaccharide from a glycosylatedsubstrate using an exo-glycosidase. In the present example, theexo-glycosidase is a β-galactosidase that cleaves the terminalβ-galactose residue to release the free monosaccharide (D-galactose)from a substrate leaving the degalactosylated aglycone.

FIG. 2. Capture of and further optional processing of a monosaccharideon solid supports using two types of capture groups. The equilibriumbetween the closed-ring form and the open-chain carbonyl of the releasedmonsoaccharide B is shown in D. The carbonyl form reacts withimmobilized —NH₂ groups on E, with loss of water, to give G. Unreacted—NH₂ groups in G can be capped to yield I, the C═N bond in I can bereduced to give J wherein the —NH group can also be capped to give L.The initial capture product G can also be reduced directly to K, whichcan be further capped to give L. A second type of solid-support Fbearing —YH capture groups can also react directly with the open-chaincarbonyl group in D, by addition to give H.

FIG. 3. Reaction of the detection agent TAG-R-Boronate (M) with thecaptured monosaccharide (any of G—L, FIG. 2) to form a covalent adductN. The abbreviated schematic for any of G—L (FIG. 2) shows that theimmobilized monosaccharide contains a diol that is capable of binding tothe boron atom. Other binding groups, such as triols, amino-alcohols ora hydroxy-acids, are also possible. The TAG can be detected eitherdirectly, or indirectly through a perturbation of the solid support.

FIG. 4. Release of bound TAG-R-Boronate (P) back into solution from theinsoluble complex N using a competing ligand O. The structure of P willdepend on the structure of the competing ligand, but will incorporateOH, OR or N groups bound to the boron atom.

FIG. 5. Examples of structures a-c of TAG-R-Boronate M.

a) Phenyl boronic acid derivatives:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO,

X-TAG substituent may be o, m or p to the boron

Example: X=m-NH, TAG=Tetramethylrhodamine;

b) Phenyl boronic acid derivatives with an o-aminometyl moiety:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO,

X-TAG substituent may be o, m or p to the boron

R═H, alkyl, cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl,substituted aryl. Example: X=p-NH, TAG=Tetramethylrhodamine, R=Me;

c) Phenyl boronic acid derivatives with a substituted o-aminomethylmoiety:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, R═H, alkyl,cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl, substitutedaryl.

Example: X═CH₂, TAG=Tetramethylrhodamine, R═H

The free acid form of the boronate is shown, though esters are alsoimplied.

FIG. 6. Examples d-f of structures of TAG-R-Boronate M.

d) Phenyl boronic acid derivatives bearing an electronegativesubstituent:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO,

X-TAG and Y substituents may be o, m or p to the boron

Y=electronegative group such as NO₂, COOR, CN, COR, SO₂OH, SO₂R,CF₃(R═H, alkyl or aryl)

Example: Y=m-NO₂, X=m-CONHCH₂CH₂NH—, TAG=Lissamine (2);

e) Pyridine boronic acid derivatives:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO,

B and X-TAG substituent may be o, m or p to ring Nitrogen

Example: X=m-NH, m-B(OH)₂, TAG=Tetramethylrhodamine;

f) Phenyl boronic acid derivatives bearing a sulfonamide or a sulfonesubstituent: S may be o, m or p to the boron

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring,

NR(R═H, alkyl, cycloalkyl, aryl, substituted alkyl, substitutedcycloalkyl, substituted aryl)

Example: X=NHCH₂CO, TAG=Tetramethylrhodamine.

The free acid form of the boronate is shown, though esters are alsoimplied.

FIG. 7. Examples g-i of structures of TAG-R-Boronate M.

g) Phenyl boronic acid derivatives bearing an o-hydroxymethyl group:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO,

X-TAG substituent may be o, m or p to the boron

Example: X=p (to CH₂)—NH, TAG=Tetramethylrhodamine (1);

h) Phenyl boronic acid derivatives bearing a substituted o-hydroxymethylgroup:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, CO

Example: X═CH₂CO, TAG=Tetramethylrhodamine;

i) Phenyl boronic acid derivatives bearing a quaternary ammoniumsubstituent:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring,

R═H, alkyl cycloalkyl, aryl, substituted alkyl, substituted cycloalkyl,substituted aryl. Example: R═CH₂CO, TAG=Tetramethylrhodamine.

The free acid form of the boronate is shown, though esters are alsoimplied.

FIG. 8. Examples j-I of structures of TAG-R-Boronate M.

j) Derivatives with multiple aryl boronic acids;

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO

Y=a linking moiety

X-TAG substituent may be on the aryl groups or the linking moiety

Example: Ar=phenyl, Y=m,m-CH₂ TAG=Tetramethylrhodamine;

k) Boronic acid derivatives of aromatic 5-membered ring heterocycles:

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO

X-TAG and B(OH)₂ substituent may be o, m

M=N, O, S

Example: X=o (to M)-CO, TAG=Tetramethylrhodamine, m (to M)-B(OH)₂.

l) Phenyl boronic acid derivatives containing fused rings.

X=alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl,substituted heteroaryl, fused alkyl or heteroaryl ring, NH, O, S, CO

X-TAG substituent may be o, m or p to the boron

Rings may be fused at any two adjacent positions

Rings may be substituted

Example: of an o-naphtalene derivative:

X=m-NH, TAG=Tetramethylrhodamine, fused ring=phenyl.

The free acid form of the boronate is shown, though esters are alsoimplied.

FIG. 9. Structures of some capping agents.

FIG. 10. Synthesis of Capture Beads

FIG. 11. Synthesis of fluorescent hydroxymethyl-boronate 1

FIG. 12. Synthesis of fluorescent nitro-boronate 2

FIG. 13. Staining of treated capture beads with 1, and elution of 1 fromstained beads with glycerol. Top panel: the beads designated AFet+ andGal-std appear bright red, the others are white. Middle panel: afterwashing with glycerol all the beads appear white. Bottom panel: theglycerol washes of beads designated AFet+ and Gal-std appear bright red,the others are clear.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Aliphatic groups: Aliphatic compounds are non-aromatic organiccompounds, in which carbon atoms are joined together in straight orbranched chains rather than in rings. Aliphatics include not only thefatty acids and other derivatives of paraffin hydrocarbons (alkanes),but also unsaturated compounds, such as ethylene (the alkenes) andacetylene (the alkynes). The most frequently found non-carbon atomsbound to the carbon chain include hydrogen, oxygen, nitrogen, sulfur,and various halides.

Alicyclic compounds such as cycloalkanes are aliphatic compounds thathave one or more non-aromatic cycles in their chemical structure.Bicycloalkanes have two rings of carbon joined at one or two carbons.Most aliphatic compounds have very exothermic combustion reactions, thusallowing hydrocarbons such as methane to fuel Bunsen burners in thelaboratory, for example.

The aliphatic residue can be an optionally substituted linear aliphaticresidue or an optionally substituted branched aliphatic residue. Thealiphatic residue can also be an optionally substituted cyclic alkyl.“Cyclic alkyl” includes groups such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such ringssubstituted with straight and branched chain alkyl groups as definedabove. also includes polycyclic alkyl groups such as, but not limitedto, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such ringssubstituted with straight and branched chain alkyl groups as definedabove. Thus, unsubstituted alkyl groups includes primary alkyl groups,secondary alkyl groups, and tertiary alkyl groups. Unsubstituted alkylgroups may be bonded to one or more carbon atom(s), oxygen atom(s),nitrogen atom(s), and/or sulfur atom(s) in a ligand. The cyclicaliphatic residue can e.g. comprise or consist of a C5-C16 cycloalkylgroup. Shorter chain lengths can also occur, typically when thecycloalkyl is substituted with an aryl or heteroaryl residue. In oneembodiment, the optionally substituted aliphatic residue comprises orconsists of a C5-C20 alkyl group. Shorter chain lengths can also occur,typically when the alkyl is substituted with an aryl or heteroarylresidue. Further examples of alkyl groups substituted with aryl orheteroaryl includes, for example, a linear (C1-C10), branched (C4-C10)or cyclic (C5-C10) group, such as a methyl group, ethyl group, propylgroup, such as a n-propyl group and an isopropyl group, butyl group,such as n-butyl group, isobutyl group, t-butyl group, n-amyl group,pentyl group, such as neopentyl group, cyclopentyl group, hexyl group,such as n-hexyl group, cyclohexyl group, heptyl group, octyl group, suchas n-octyl group, nonyl group, such as n-nonyl group, decyl group, suchas n-decyl group, undecyl group, dodecyl group, mentyl group,2,3,4-trimethyl-3-pentyl group, 2,4-dimethyl-3-pentyl group, and thelike.

Two preferred aliphatic groups are an ethyl or a methyl group.

Aromatic groups: The term “aromatic” or “aryl” moiety means either amono- or polycyclic hydrocarbon group, which has a cyclic, delocalized(4n+2) pi-electron system, including arenes and their substitutionproducts. Examples of suitable aromatic moieties for use in the presentinvention include, but are not restricted to, benzene, naphthalene,toluene, thiophene and pyridine.

Carbohydrate: The generic term ‘carbohydrate’ includes monosaccharides,oligosaccharides and polysaccharides as well as substances derived frommonosaccharides by reduction of the carbonyl group (alditols), byoxidation of one or more terminal groups to carboxylic acids, or byreplacement of one or more hydroxy group(s) by a hydrogen atom, an aminogroup, a thiol group or similar heteroatomic groups. It also includesderivatives of these compounds.

Glycose: “Glycose” in the present context refers to a monosaccharide,which can for example be either an aldose (a polyhydroxy aldehyde), aketose (a polyhydroxy ketone), an oxidized derivative thereof includingan alduronic acid (e.g. glucuronic acid), ketoaldonic acid (e.g. sialicacid or Kdo) as well as deoxy-derivatives and amino-derivatives thereof.

Sugar: The term “sugar” as used herein covers monosaccharides,oligosaccharides, polysaccharides, as well as compounds comprisingmonosaccharide, oligosaccharide, or polysaccharide. The terms“carbohydrate” and “sugar” are herein used interchangeably.

Oligo/poly-saccharide: Oligosaccharides and polysaccharides arecompounds consisting of monosaccharides linked glycosidically. Ingeneral polysaccharides comprise at least 10 monosaccharide residues,whereas oligosaccharides in general comprise in the range of 2 to 20monosaccharides. Oligosaccharides and polysaccharides may be linear orbranched.

TAG: The term “TAG” in the present context, and in FIG. 1 (vide infra)is meant to indicate any atom, molecule or entity that can becomecovalently attached to another molecule thereby labelling said anothermolecule as having undergone the covalent attachment.

Monosaccharide: Parent monosaccharides are polyhydroxy aldehydesH—[CHOH]_(n)—CHO or polyhydroxy ketones H—[CHOH]_(n)—CO—[CHOH]_(m)—Hwith three or more carbon atoms. The generic term ‘monosaccharide’ (asopposed to oligosaccharide or polysaccharide) denotes a single unit,without glycosidic connection to other such units. It includes aldoses,dialdoses, aldoketoses, ketoses and diketoses, as well as deoxy sugarsand amino sugars, and their derivatives, provided that the parentcompound has a (potential) carbonyl group. Preferred examples ofmonosaccharides comprise in the range of 4 to 9 carbons, for example forpolyhydroxy aldehydes n is an integer in the range of 3 to 8 and forpolyhydroxyketones n+m is an integer in the range of 3 to 8. The term“Monosaccharide” can also include monosaccharide derivatives, such asthose obtained by oxidation, deoxygenation, replacement of one or morehydroxyl groups by preferably a hydrogen atom, an amino group or thiolgroup, as well as alkylation, acylation, sulfation or phosphorylation ofhydroxy groups or amino groups. Various categories of monosaccharides,all of which are envisaged as identifiable using the methods of thepresent invention, are described below:—

-   Monosaccharides with an aldehydic carbonyl or potential aldehydic    carbonyl group are called aldoses; those with a ketonic carbonyl or    potential ketonic carbonyl group, ketoses. The term ‘potential    aldehydic carbonyl group’ refers to the hemiacetal group arising    from ring closure. Likewise, the term ‘potential ketonic carbonyl    group’ refers to the hemiketal structure.-   Cyclic hemiacetals or hemiketals of sugars with a five-membered    (tetrahydrofuran) ring are called furanoses, those with a    six-membered (tetrahydropyran) ring pyranoses.-   Monosaccharides containing two (potential) aldehydic carbonyl groups    are called dialdoses)-   Monosaccharides containing two (potential) ketonic carbonyl groups    are termed diketoses-   Monosaccharides containing a (potential) aldehydic group and a    (potential) ketonic group are called ketoaldoses.-   Monosaccharides in which an alcoholic hydroxy group has been    replaced by a hydrogen atom are called deoxy sugars-   Monosaccharides in which an alcoholic hydroxy group has been    replaced by an amino group are called amino sugars. When the    hemiacetal hydroxy group is replaced, the compounds are called    glycosylamines.-   The polyhydric alcohols arising formally from the replacement of a    carbonyl group in a monosaccharide with a CHOH group are termed    alditols.-   Monocarboxylic acids formally derived from aldoses by replacement of    the aldehydic group by a carboxy group are termed aldonic acids.-   Oxo carboxylic acids formally derived from aldonic acids by    replacement of a secondary CHOH group by a carbonyl group are called    ketoaldonic acids.-   Monocarboxylic acids formally derived from aldoses by replacement of    the CH2OH group with a carboxy group are termed uronic acids.-   The dicarboxylic acids formed from aldoses by replacement of both    terminal groups (CHO and CH2OH) by carboxy groups are called aldaric    acids.

Glycosides: Glycosides are mixed acetals formally arising by eliminationof water between the hemiacetal or hemiketal hydroxy group of a sugarand a hydroxy group of a second compound. The bond between the twocomponents is called a glycosidic bond.

Method for Analysis of a Terminal Monosaccharide on a GlycosylatedSubstrate

In a first aspect of the present invention is provided a method foranalysis of a terminal monosaccharide on a glycosylated substrate, saidmethod comprising the steps of:—

-   (i) detaching said monosaccharide from said glycosylated substrate,    preferably using an exoglycosidase;-   (ii) allowing said detached monosaccharide to covalently bind to a    capture group on a solid support;-   (iii) incubating said covalently bound monosaccharide with a    detection agent with formula X:

TAG-R-Boronate

wherein TAG=a tag moiety capable of being detected

-   -   R=organic moiety    -   Boronate=a boronic acid moiety or ester thereof,        said boronate being attached to a carbon atom comprised in said        R group;

-   (iv) allowing the detection agent to bind the monosaccharide

-   (v) detecting detection agent having bound to the monosaccharide.

In one embodiment of the invention it is preferred that the above methoddoes not comprise a step of reducing the bond between the capture groupand the monosaccharide after immobilisation to the solid support. Thus,for example it is preferred that if the monosacchride is bond to thesolid support via a C═N linkage, then said C═N is not intentionallyreduced by contacting the immobilised monosaccharide with a reducingagent such as with a borane or a borohydride.

In another embodiment it is preferred that the immobilisedmonosaccharide is not contacted with a reducing agent before orsimultaneously with incubation with the detection agent.

The stages of the method are described in more detail herein below:—

-   (i) detaching said monosaccharide from said glycosylated substrate

Stage (i) of the method of the present invention comprises the step ofdetaching a monosaccharide from a glycosylated substrate A (see e.g.FIG. 1)

In one embodiment of the invention it is preferred that saidglycosylated substrate is not an immobilised sugar of the generalstructure Sugar-C═N-linker-Spacer-Solid.

Monosaccharide: In a preferred embodiment, the monosaccharide is anaturally occurring monosaccharide or a monosaccharide which has beenliberated from a naturally occurring or recombinantly produced compoundcomprising a carbohydrate, preferably without having been subject tofurthermore modifications after liberation.

In one preferred embodiment of the present invention, the monosaccharideis a glycose, such as selected from the group consisting of: an aldose,a ketose, a deoxy sugar, an amino sugar,

and derivatives thereof, such as an oxidized derivative thereof.

Examples of suitable glycoses include galactose, fucose,N-acetylglucosamine, galacturonic acid, and sialic acid.

Any glycosylated substrate comprising monosaccharides that is known toone skilled in the art can be used in the methods of the presentinvention. For example, said glycosylated substrate can be selected fromthe group consisting of: a glycosylated antibiotic, a glycoprotein, aglycolipid, a glycosylated steroid, an oligosaccharide, apolysaccharide.

In one preferred embodiment of the present invention, the glycosylatedsubstrate is a substrate obtainable from a eukaryotic organism. Thus,the glycosylated substrate can be selected from the group consisting of:a glycoprotein, a glycolipid or a proteoglycan; preferably derived froma eukaryotic organism. In another embodiment, said glycosylatedsubstrate is selected from the group consisting of: a glycoprotein, aglycolipid or a proteoglycan, preferably obtainable or obtained from thecell membrane of a eukaryotic cell.

It can also be desirable to carry out the method of the presentinvention on a glycosylated substrate obtainable from a prokaryoticorganism. Thus, in one embodiment of the present invention, theglycosylated substrate is selected from the group consisting of: aglycoprotein, a glycolipid, a lipo-polysaccharide, or a polysaccharide,obtainable or obtained from a prokaryotic organism. In anotherembodiment, the glycosylated substrate is selected from the groupconsisting of: a glycoprotein, a glycolipid, a lipo-polysaccharide, or apolysaccharide obtainable from or obtained from the cell surface ormembrane of a prokaryotic organism.

In another embodiment, the glycosylated substrate is selected from thegroup consisting of: a glycosylated antibiotic, a glycosylated steroid,a glycosylated natural product or a glycosylated peptide.

Glycosylated substrates may be derived from a variety of sources. Forexample the glycosylated substrate may be obtained from a livingorganism or part of a living organism, such as animals or plants or fromone or more specific animal or plant tissues, from organisms such asprokaryotic or eukaryotic cells, from viruses, from in vitro cultivatedmammalian cells, insect cells, plant cells, fungi, bacterial cells,yeast, or phages. For example the glycosylated substrate may be isolatedfrom extracts of any of the aforementioned cells, microbial organisms orliving organisms. Such extracts may comprise glycosylated substrates,such as free carbohydrates. Extracts may also comprise compoundscomprising monosaccharide, oligosaccharide, polysaccharide orcarbohydrate moieties, notably glycoproteins or glycolipids or smallorganic molecules to which carbohydrates are attached, which aregenerally referred to as glycosides. Glycoproteins are compounds inwhich a carbohydrate component is linked to a peptide, polypeptide orprotein component. Thus as used herein the term glycoprotein also coverproteoglycans and glycosaminoglycans. Glycolipids are compoundscontaining one or more monosaccharide, oligosaccharide, polysaccharideor carbohydrate moieties bound by a glycosidic linkage to a hydrophobicmoiety such as an acylglycerol, a sphingoid, a ceramide(N-acylsphingoid) or a prenyl phosphate. Glycosides can for example besmall (MWt 100-5000) organic molecules glycosidically linked to one ormore sugars via either O, N or S.

Glycosylated substrates may also be the products of chemical synthesis,or chemical/enzymatic synthesis, such as oligosaccharides prepared invitro by chemical synthesis in solution or on the solid phase. Thesesame synthetic oligosaccharides may be further modified by enzymaticreaction, such as for example by the sulfation, phosphorylation orglycosylation. Thus the methods described herein may also be used foridentification of monosaccharides of synthetic or semi-syntheticoligosaccharides or oligosaccharide libraries.

Optionally, the glycosylated substrate to be analysed can be comprisedwithin a sample—for example, a complex sample, such as a samplecomprising non-glycosylated substrates. It can thus be beneficial insome embodiments that the method of the present invention comprises anadditional step after step (i), wherein (preferably high molecularweight) components of said sample are removed, such as for removal ofmacromolecules, such as larger polysaccharides or non-polysacharidecomponents, particularly polypeptides. One method of removing saidcomponents is by size exclusion, preferably by ultrafiltration or bydialysis. Another method of removing the said components involvedpassing the sample through a membrane that is permeable to themonosaccharide but not to molecules with high molecular weights, such asproteins. Said membrane can for example be a Centricon (Millipore)membrane or a dialysis membrane. In another embodiment, said componentsof the sample are removed by absorption on hydrophobic phases such asC18 or carbon.

Detachment step:—The detachment step of step (i) can be carried out by arange of methods known to one skilled in the art. Preferably, saiddetachment step is carried out using an exo-glycosidase, such as abacterial exo-glycosidase, such as for example an exo-glycosidaseselected from the group consisting of:

alpha-mannosidase, alpha-glucosidase, alpha-galactosidase,alpha-xylosidase, alpha-fucosidase, alpha-N-acetylglucosaminidase,alpha-N-acetylgalactosaminidase, alpha-glucuronidase, alpha-iduronidase,alpha-sialidase, beta-mannosidase, beta-glucosidase, beta-galactosidase,beta-xylosidase, beta-fucosidase, beta-N-acetylglucosaminidase,beta-N-acetylgalactosaminidase, beta-glucuronidase, beta-iduronidase andbeta-sialidase.

Thus, said terminal monosaccharide can be detached using glycosidasesacting on bacterial polysaccharides or glycolipids, cleaving deoxysugars, amino sugars, substituted amino sugars, branched chain sugars,O-methyl sugars and the like.

Many useful glycosidases are further described in the art, for exampleany of the glycosidases described in U.S. Pat. No. 5,100,778 or WO92/19974 may be employed with the present invention.

An example of the release of a terminal monosaccharide from aglycosylated substrate using an exo-glycosidase is shown in FIG. 1:—

The soluble substrate monosaccharide-O—R-Substrate (A) is incubated withan exo-glycosidase of known specificity in a solution in which theglycosidase is soluble, typically a buffer solution with or withoutadded cations. The exo-glycosidase is capable of cleaving a singleterminal monosaccharide residue from a glycoside. The R group in A ispreferably a carbohydrate (such as a monosaccharide or oligosaccharide)unit or an amino acid in glycoproteins or glycopeptides, a glycose unitin polysaccharides or oligosaccharides, a glycose unit or a lipid inglycolipids, or a glycose unit or an organic aglycone in e.g.glycosylated antibiotics or steroids. If the terminal monosaccharideunit has a structure and anomeric configuration (either α or β) that ishydrolysable by the glycosidase, it is cleaved to produce a reducingsugar (monosaccharide, B) and the aglycone (HO—R-substrate, C).

The specificities of many glycosidases are known, and suchwell-characterized glycosidases have been used in the sequencing ofoligosaccharides. Thus, these enzymes can be specific for thestereochemistry of the monosaccharide-rings and the α or β configurationof the glycosidic linkages. Many of them are also specific with respectto the exact position of attachment of the glycose to the next sugar.For example, some α-glycosidases cleave only α1-3 linkages and othersonly α1-6 linkages. Thus, the entire structure of an an oligosaccharidecan sometimes be determined by successive glycosidase digestion.

In another embodiment, the terminal monosaccharide is detachedchemically, such as for example using acid hydrolysis, such as mild acidhydrolysis. For mild hydrolysis one can, for example, treat theglycosylated substrate with 0.1 N aq. trifluoroacetic acid at 80 degreesCelsius for 1 h. O-linked monosaccharides may be cleaved fromglycoproteins by chemical methods, such as alkaline β-elimination orenzymatically using enzymes such as an O-glycosidase. Monosaccharidesmay also be cleaved from small organic molecules using either acidic orbasic reactions.

(ii) Allowing Said Detached Monosaccharide to Covalently Bind to aCapture Group on a Solid Support

Stage (ii) of the method of the present invention comprises the step ofallowing said detached monosaccharide to covalently bind to a capturegroup on a solid support.

Capture group:—By “capture” group is meant a reactive chemical groupcapable of making a covalent bond by nucleophilic attack at the carbonylgroup of the liberated monosaccharide (B). Examples of two differentversions of this “capture” step are illustrated in FIG. 2:—Theequilibrium between the closed-ring form and the open-chain carbonyl ofthe released monosaccharide B is shown in D. The carbonyl form reactswith immobilized (e.g. NH₂)-capture groups linked to the solid support(E), to give a captured monosaccharide (G). Unreacted capture groups inG can optionally be capped to yield “capped” capture groups (I),optionally with further reducing and/or capping steps, to give J and L.The initial capture product G can also be reduced directly to K, whichcan be further capped to give L. In an alternative embodiment, a secondtype of solid-support F bearing —YH capture groups can also reactdirectly with the open-chain carbonyl group form of the monosaccharidein D, by addition to give a monosaccharide captured as H. Preferably,the covalent adduct formed between the monosaccharide bound to thecapture group on the solid support comprises at least two OH— groups.

Many suitable capture groups for use in the method of the presentinvention are known by one skilled in the art. Preferably, the capturegroup comprises or consists of an —NHR group, where R is selected fromthe group consisting of: an alkyl, aryl, substituted alkyl orsubstituted aryl group. For example, the capture can comprise or consistof an —NH₂ group. In one preferred embodiment, the capture groupcomprises or consists of the structure -M-NH₂, wherein M is aheteroatom.

In one embodiment, the capture group comprises or consists of a sulphuratom or phosphorous atom.

In another preferred embodiment, the capture group comprises or consistsof an acidic —CH— group capable of ionization to a carbanion ansubsequent nucleophlic attack at the sugar aldehyde. Compounds of thistype are known by one skilled in the art and typically include a CHgroup adjacent to one or more of the follow groups:—

one or more carbonyl groups (—CH—CO—),

a nitrile group (giving e.g. —CH—CN)

a nitro group (giving e.g. —CH—NO2)

a sulfone group (giving e.g. —CH—SO2—)

Capture groups comprising at least one —NH₂ group: According to onepreferred embodiment of the present invention the linker comprises acapture group, wherein the capture group comprises at least one —NH₂group. In a favourable format, the capture group terminates in an —NH₂group that is attached to the linker through an optional group R. Thusthe capture group preferably is of the structure R—NH₂. R may be asimple alkyl, aryl or substituted alkyl or aryl group. Preferably, Rshould contain a heteroatom directly attached to the —NH₂ group, toproduce structures of the type linker-M-NH₂, wherein M is a heteroatom(i.e. not carbon), preferably M is selected from the group consisting ofN, O and S. Especially favourable are compounds where M is a heteroatom,such as in the structures linker-O—NH₂, linker-NH—NH₂, linker-CO—NH—NH₂,linker-NH—CO—NH—NH₂, linker-S(O)₂NH—NH₂ and linker-S—NH₂.

In this embodiment, the capture of the monosaccharide is done byreacting the —NH₂ group of the capture group with e.g. the reducing endof said monosaccharide, i.e. with the aldehyde, ketone or hemiacetalgroup. The reaction can occur at any pH values but is most favored inthe range of pH 2-9. The methods may involve the addition of one or moreadditives, such as additives which may either facilitate or favourablyalter the equilibrium between the open chain aldehyde form of themonosaccharide and the hemiacetal form of the monosaccharide, whereinthe open chain aldehyde form is preferred. The additive may for examplebe metal ions, boronates or silicates. The capture produces a speciesattached to the solid support through a covalent double bond (shown asC═N) where the C is derived from the monosaccharide moiety and N fromthe capture group. This immobilized monosaccharide may also be inequilibrium with its cyclic ring form, in particular if themonosaccharide was a pyranose, then the immobilised monosaccharide maybe in equilibrium with its cyclic 6-membered ring form (see for examplecompounds B and D of FIG. 2), but it may also be in equilibrium with its5-membered ring form if the appropriate OH group on the monosaccharideis unsubstituted.

-   Linker group: The capture group can optionally be attached to the    solid support via a linker group. Said linker group can be any    molecule capable of linking the capture group to the solid support.    The linker may be any of a large variety of linkers such as those in    common use in solid-phase organic synthesis. The linker may either    be a non-cleavable linker or a cleavable linker.

Non-cleavable linkers may for example be alkyl, aryl, ethers or amides,wherein any of the aforementioned may optionally be substituted. Forexample any of the aforementioned may be substituted with heteroatoms orthey may contain, O-alkyl,alkyl, aryl or heteroatoms as branches. In oneexample the linker comprises or essentially consists of PEG and/orpolyamide.

The linker may comprise a site where a reaction can be made to occur tosever the part containing the capture group (including the molecules ithas captured and which have been optionally further modified) from thesolid support. Such linkers are referred to as cleavable linkers, andare in wide use in solid phase organic synthesis. Examples of cleavablelinkers are known where the cleavage can be effected by electrophiles,nucleophiles, oxidizing agents, reducing agents, free radicals, acid,base, light, heat or enzymes.

Cleavable linkers may for example be acid labile (for example, the Rinkamide as described in Rink, 1987, Tetrahedrom Lett., 28: 387 andtraceless silyl linkers as described in Plunkett et al., 1995, J. Org.Chem., 60: 6006-7), base labile (for example, HMBA as described inAtherton et al. 1981, J. Chem. Soc. Perkin Trans, 1: 538), orphotolabile (for example, 2-nitrobenzyl type as described in Homles etal., 1995, J. Org. Chem., 60: 2318-2319). The linkers may be morespecific and restrictive of the type of chemistry performed, such assilyl linkers (for example, those cleaved with fluoride as described inBoehm et al., 1996, J. Org. Chem., 62: 6498-99), allyl linkers (forexample, Kunz et al., 1988, Angew. Chem. int. Ed. Engl., 27: 711-713),and the safety catch sulfonamide linker (for example, as described inKenner et al., 1971, Chem. Commun., 12: 636-7). Enzyme cleavable linkersmay for example be any of the enzyme cleavable linkers described inReents et al., 2002, Drug Discov. Today, 7: 71-76, or any functionalisedderivatives of the enzyme-labile protecting groups described in thereview by Waldmann et al., 2001, Chem. Rev. 101: 3367-3396. Heat labilelinkers may for example be of the type described in Meng et al., 2004,Angew. Chem. Int. Ed., 43: 1255-1260.

-   Reaction conditions: The capture reaction may be performed in any    useful solvent. A person of ordinary skill in the art will readily    be able to identify a useful solvent for any given compound. The    solvent may for example be selected from the group consisting of    water, aqueous buffer, organic solvents and mixed aqueous and    organic solvents. The solvent may also be any of the aforementioned    comprising one or more additives such as acids, bases, organic    amines, anilines, salts, divalent metal cations, detergents,    complexing agents including inclusion-complex-forming molecules such    as cyclodextrins or calixarenes, chelating agents (for example    EDTA), borates, boronates or silicates.

In a preferred embodiment the amount of solid support added to thereaction is adjusted so that a molar excess of capture groups arepresent in relation to the monosaccharide, preferably said excess islarge, such as at least 2 times, preferably at least 5 times, morepreferably at least 10 times, such as at least 50 times, for example atleast 100 times or more. This excess will ensure a more efficientcapture of the monosaccharide.

The capture reaction may be carried out at any temperature, butpreferably at temperatures in the range of 0 to 100° C.

-   Solid support: Any suitable solid support capable of supporting the    capture groups and known to one skilled in the art can be used in    the methods of the present invention. For example, the solid support    can be selected from the group consisting of polymers, solids,    insoluble particles and surfaces. Examples include PEGA and SPOCC.

The solid support is preferably a bead. In another preferred embodiment,the solid support is a slide, such as a glass slide, a microtiter well,or a metal-coated slide, for example a gold-coated slide. The solidsupport can be a hydroxylamine-modified surface, in which case it ispreferably a controlled pore glass (CPG) bead or glass slide.

The term “solid support” as used herein covers physical solids as wellas insoluble polymers, insoluble particles, surfaces, membranes andresins, preferably the solid support is an insoluble polymer, aninsoluble particle, a surface or a resin.

Thus the “solid support” may be an insoluble inorganic matrix (such asglass), an insoluble polymer (such as a plastic, for examplepolystyrene), an insoluble matrix consisting of parts of both organicand inorganic components (e.g. some hybrid silicates, such as compoundsof the structure R—Si—O—), organic polymers in common use in solid-phasesynthesis (polystyrenes, PEGA resins, PEG resins, SPOCC resins andhybrids thereof), polyethylene glycol chains (which can be soluble incertain organic solvents and made insoluble by the addition of othersolvents). The solid may also be a metal (such as gold), an alloy, or acomposite such as for example indium-tin oxide or mica.

Organic polymers used in solid-phase synthesis for example include:TentaGel (commercially available from Rapp polymere, Tübingen, Germany),ArgoGel (commercially available from Argonaut Technologies Inc., SanCarlos, Calif.), PEGA (commercially available from Polymer Laboratories,Amherst, Mass.), POEPOP (Renil et al., 1996, Tetrahedron Lett., 37:6185-88; available from Versamatrix, Copenhagen, Denmark) and SPOCC(Rademann et al, 1999, J. Am. Chem. Soc., 121: 5459-66; available fromVersamatrix, Copenhagen, Denmark).

In one embodiment of the invention the solid support is a sensor, suchas a surface acoustic wave sensor (such as any of the sensors describedin Samoyolov et al. 2002, J. Molec. Recognit. 15: 197-203), a surfaceplasmon resonance sensor (such as any of the sensors reviewed by Homolaet al., 1999, Sensors and Actuators B, 54: 3-15), or a nanomechanicalcantilever sensor such as described by Mukhopadhyay in Nano Lett. 2005,5, 2385-88.

Such solid supports may be inorganic materials such as glass, metalssuch as gold, organic polymeric materials or hybrids thereof and may becovered various coatings such as proteins or polysaccharides, oligomerssuch as as dendrimers or polymers such as polyacrylamide or polyethyleneglycol.

In a preferred embodiment the solid support is glass.

-   Washing: Once the terminal monosaccharide has been immobilised on    the solid support through reaction with the capture group (see e.g.    steps exemplified in FIG. 2), the solid supports can optionally be    washed to remove non-covalently bound material. Accordingly, if the    glycosylated substrate is, for example, provided in a sample    comprising other compounds, the reducing sugar may be purified from    said sample. It is thus comprised within the present invention that    the glycoslated substrate is provided in a non-purified form.

The skilled person will readily be able to identify suitable washingconditions for a given immoblised monosaccharide (e.g. any of compoundsG-L in FIG. 2). The washing may for example be done with any of theherein-mentioned solvents optionally comprising any of theherein-mentioned additives in addition to detergents and denaturingagents. The washing may be performed at any temperature, but preferablyat temperatures in the range of 0-100° C.

(iii) Incubating Said Covalently Bound Monosaccharide with a DetectionAgent

Stage (iii) of the method of the present invention comprises the step ofincubating said covalently bound monosaccharide with a detection agentwith formula X:

TAG-R-Boronate

wherein TAG=a tag moiety capable of being detected

-   -   R=organic moiety    -   Boronate=a boronic acid moiety or ester thereof,        said boronate being attached to a carbon atom comprised in said        R group. Preferably, an excess of said detection agent is used.

By “Boronate” is meant any suitable chemical moiety comprising a boronicacid moiety, or an ester thereof, preferably the boronate is boronicacid.

The detection agent can for example comprise aryl boronate orheteroarylboronate. The process is shown schematically in FIG. 3.

It is preferred that the detection agent has a formula selected from thegroup consisting of:—

TAG-R—B(OH)₂, TAG-R—B(OH)(OR′) or TAG-R—B(OR′)(OR″)

wherein R′ and R″ may be either aliphatic or aromatic and are optionallycovalently attached to R, for example resulting in a cyclic boronatestructure. For example R′ and R″ may individually be selected from thegroup consisting of C₁₋₆ linear or branched alkyl, C₅₋₇ aliphatic ringand C₅₋₇ aromatic ring. In the event R is a ring, R′ may also form afused ring with R, and in this embodiment R′ may be a 4-7 memberedaliphatic heterocycle, which is optionally substituted. For example,said heterocycle may be a 5 membered cycle including the B and the Oatom to which R′ is attached. Said heterocycle may not be substituted orit may be substituted.

Thus, in one embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative, such as as exemplified bygroup a) in FIG. 5.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative with an o-aminomethyl moietysuch as as exemplified by group b) in FIG. 5.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative with a substitutedo-aminomethyl moiety, such as as exemplified by group c) in FIG. 5.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative bearing an electronegativesubstituent, such as as exemplified by group d) in FIG. 6.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative with a sulfonamide or asulfone substituent, such as as exemplified by group e) in FIG. 6.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a pyridine boronic acid derivative, such as as exemplified bygroup f) in FIG. 6.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative bearing an o-hydroxymethylgroup, such as as exemplified by group g) in FIG. 7.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative bearing a substitutedo-hydroxymethyl group, such as as exemplified by group h) in FIG. 7.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative bearing a quaternary ammoniumsubstituent, such as as exemplified by group i) in FIG. 7.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Multiple aryl boronic acid derivative, such as asexemplified by group j) in FIG. 8.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Boronic acid derivative of an aromatic 5-membered ringheterocycle, such as as exemplified by group k) in FIG. 8.

In another embodiment of the present invention, the TAG-R-Boronatecomprises a Phenyl boronic acid derivative with 2 or more fused rings,such as 2, 3, 4, 5 or 6 rings, such as as exemplified by group I) inFIG. 8.

In one preferred embodiment, the detection agent has the formula of thetetramethylrhodamine-derived fluorescent hydroxymethyl-boronate 1 shownin FIG. 11.

In another preferred embodiment, the detection agent is according to theformula of the lissamine-derived fluorescent nitro-boronate 2 shown inFIG. 12.

The R moiety can be any chemical group, such as an aliphatic or aromaticmoiety, as described herein. Thus, R may for example be an aromaticcycle or heteroaromatic cycle, such as a 5 to 6 membered aromatic orheteroaromatic cycle or a fused ring aromatic ring system, for example 2fused rings of each 5 to 6 members fused at any two positions (see e.g.FIG. 8, I). For example, the heteroaromatic cycle may be a 5 to 6membered ring, such as a 5 membered ring comprising in the range of 1 to3, such as 1, for example 2 heteroatoms selected from the groupconsisting of N, O and S. Said aromatic cycle or heteroaromatic cyclemay optionally be substituted, for example substituted with alkyl, suchas C₁₋₆ alkyl, amino alkyl, such as C₁₋₆ amino alkyl, for exampleC—NR^(x) ₂, a primary or secondary or tertiary amino group or sulphate,wherein R^(x) may be —H, alkyl, cycloalkyl, aryl, substistuted alkyl,substitued cycloalkyl or substituted aryl, such as —H, C₁₋₆ alkyl, C₅₋₇cycloaryl or C₅₋₇ aryl.

In a preferred embodiment said aromatic or heteroaromatic cycle issubstituted with X-TAG, or one of aforementioned substiuents arecovalently attached to X-TAG, wherein X is selected from the groupconsisting of —NH—, —CO—, alkyl (such as C1-6 alkyl, for example methylor ethyl), aryl (such as C5 to 7 aryl), heteroaryl (such as a 5 to 7membered aryl cycle), substituted alkyl, substituted aryl, substitutedheteroaryl, fused alkyl or heteroalkyl, heteroaryl ring, —O— and —S—.For example, X may be selected from the group consisting of —NH—, —CO—,—CH₂—, —CH₂—CH₂—, phenyl, —O— and —S—.

It may be desirable to release the detection agent from the solidsupport either before or after the detection step, for example toimprove quantification of certain types of detection agent. However, insome embodiments of the invention the detection agent is not releasedfrom the solid support. The release step may for example be effected bycontacting the solid support with a solution of a soluble compound thatcompetes with the detection agent for binding of the monosaccharide, asshown in FIG. 4.

Said soluble compound can, for example, be a polyalcohol such asglycerol or glucitol, or an amino-alcohol such as diethanolamine.

TAG moiety:—The detection agent is preferably provided comprising a TAGmoiety. Any suitable TAG moiety can be used, such as e.g. selected fromthe group consisting of: a fluorescent moiety, a luminescent moiety, anda coloured moiety. Suitable specific moieties include, inter alia,coumarin, tetramethylrhodamine, lissamine or fluorescein.

In one preferred embodiment of the invention the TAG has beneficialspectroscopic properties. By beneficial spectroscopic properties ismeant that the TAG can easily be visualised, for example byspectrometry. Thus the TAG may for example be spectroscopicallydetectable. In a preferred embodiment the TAG is a fluorescent TAG.Examples of such TAGs can be found in the Handbook of Fluorescent Probesand Research Products, by RP Haugland, 9^(th) Ed., Molecular Probes.

The product of addition of such a TAG can absorb and re-emit light thatcan be detected. The number of such TAGs present on the solid supportwill reflect the number of monosaccharides captured on the solidsupport. The number of monosaccharides originally present in a samplecan therefore be estimated by the fluorescence of the TAG, provided thatthe provided solid supports comprise an excess of capture groups. TAGsother than fluorescent molecules can also be used. These can includeradioactive TAGs, phosphorescent TAGs, chemiluminescent TAGs,UV-absorbing TAGs, nanoparticles, quantum dots, coloured compounds,electrochemically-active TAGs, infrared-active TAGs, TAGs active inRaman spectroscopy or Raman scattering, TAGs detectable by atomic forcemicroscopy or TAGs comprising metal atoms or clusters thereof.

If the solid support is a sensor, such as a surface acoustic wavesensor, a surface plasmon resonance sensor, or a cantilever, thenaddition of such a species that binds specifically to the TAG can resultin the production of a signal that is proportional to the TAG andtherefore to the number of sugar molecules. An example is when the TAGis a biotin residue, commonly introduced by reaction with an activeester of biotin. Addition of an avidin-protein, when the TAG is a biotinresidue, can result in signal that is readily detected and reported bythe sensor. Other examples of sensors that can be used to detect thebinding of second binding partners to immobilized TAGs include but arenot limited to piezoelectric sensors, amperometric sensors, surfaceplasmon fluorescence spectroscopy sensors, dual polarizationinterferometry (DPI) sensors, wavelength-interrogated optical sensors(WIOSs), impedence sensors, optical waveguide grating coupler sensors,acoustic sensors and calorimetric sensors.

Once the monosaccharide has been attached to a TAG with spectroscopicproperties, then said spectroscopic properties may be determined. Theoptical properties may be determined for sugars still immobilised on thesolid support (such as for compound N of FIG. 3) or for sugars releasedto solution by a competing ligand (for example for compound P of FIG.4). Depending on the nature of the TAG with spectroscopic properties,said properties may be determined using conventional methods, such asspectrometry. Thus the methods of the invention may comprise the step ofdetecting the TAG attached to the monosaccharide by spectrometry.

In one embodiment of the invention it is preferred that the TAG is acoloured moiety or a fluorescent moiety, which may even be detectable byvisual inspection by eye. In this the embodiment the TAG may for examplebe any fluorescent moiety known to the skilled person, for example anyof the fluorescent moieties described in The Handbook—A Guide toFluorescent Probes and Labeling Technologies 10^(th) edition availablefrom Invitrogen—Molecular Probes. For example the TAG may be 6-TRITC ortetramethylrhodamine.

Further preferred boronic acid compounds suitable for use in thedetection group of the present invention are disclosed in the followingreferences:

-   -   “Boronic Acids: Preparation, Applications in Organic Synthesis        and Medicine”, ed. Dennis G. Hall, pub. Wiley-VCH, in particular        in chapters 12 and 13 (“Boronic Acid-based receptors and sensors        for saccharides” and “Biological and medicinal applications of        boronic acids”).    -   Yan et al., “Boronolectins and Fluorescent Boronolectins: An        Examination of the Detailed Chemistry Issues Important for the        Design”, Medicinal Reseach Reviews, Vol. 25, No. 5, 490-520,        2005    -   Mulla et al., “3-Methoxycarbonyl-5-nitrophenyl boronic acid:        high affinity diol recognition at neutral pH”, Bioorganic &        Medicinal Chemistry Letter 14 (2004) 25-27    -   Dowlut et al., “An Improved Class of Sugar-Binding Boronic        Acids, Soluble and Capable of Complexing Glycosides in Neutral        Water”, J. Am. Chem. Soc. 2006, 128, 4226-7    -   Hoeg-Jensen., “Preparation and Screening of Diboronate Arrays        for Identification of Carbohydrate Binders”, QSAR Comb., Sci.        2004, 23    -   Boduroglu et al., “A colorimetric titration method for        quantification of millimolar glucose in a pH 7.4 aqueous        phosphate buffer”, Bioorganic & Medicinal Chemistry Letter        15 (2005) 3974-3977    -   Davis et al., “Simple and Rapid Visual Sensing of Saccharides”,        Organic Letter, 1999 Vol. 1, No. 2, 331-334    -   He et al., “Chromophore Formation in Resorcinarene Solutions and        the Visual detection of Mono- and Oligosaccharides”, J. Am.        Chem. Soc. 2003, 124, 5000-5009    -   Gray et al., “Specific sensing between inositol epimers by a        bis(boronate)”, Bioorganic & Medicinal Chemistry Letters        15 (2005) 5416-5418)

-   Capping and reduction: After binding of the terminal    monosaccharide(s) to the capture group(s), the solid support coupled    to the immobilised monosaccharide (such as compound G-L of FIG. 3)    may still contain unreacted free capture groups and can be subjected    to unique manipulations that increase the scope of its utility.

Thus, in one embodiment, a capping agent is bound to unbound capturegroups before step (iv) of the method. For example, in one preferredembodiment of the invention, subsequent to immobilisation of thereducing sugar, unreacted capture groups are capped by a capping agent,under conditions where the bound monosaccharide is preferably notreleased form the capture group on the solid support. After capping thesolid support will no longer comprise any free capture groups, but onlycapped groups of reduced reactivity towards e.g. electrophiles.

Any suitable capping agent known to one skilled in the art may be usedin the present invention. For example, said capping agent is a cappinggroup capable of reacting with a —NH₂ group. Examples of preferredcapping agents are disclosed in FIG. 9.

-   Capping groups reacting with —NH₂ groups: In one preferred    embodiment of the invention, subsequent to immobilisation of the    monosaccharide, unreacted —NH₂ groups are capped by a capping agent,    such as an acylating agents (e.g. acetic anhydride) or other    nitrogen-reactive agents well known in the art, under conditions    where the C═N bond of C does not react. After capping the solid    support will no longer comprise any free amine groups, but only    capped nitrogen atoms (N(H)CAP) of very low reactivity towards    electrophiles. The product of the capping of compound C can for    example contain an —R—N(H)CAP group, wherein the (H) may or may not    be present depending on the structure of the CAP group.

For example, in specific embodiments if the C═N bond linking the sugarto the solid support is reduced to an —NH—, it can be a formallySP³-hybridized nitrogen atom in the sequence R—NH—CH₂—. Specificreactions may be directed to this group, allowing specific andstoichimetric reactions at the monosaccharide.

Preferably the capping agent specifically reacts with the remaining —NH₂groups, without substantially reacting with the C═N functionality. Suchreagents are well known in the art an include common acylating agentsused for amid bond formation, e.g. acetic anhydride, other alkanoic acidanhydrides, aromatic anhydrides (e.g. benzoic anhydride), cyclicanhydrides (e.g. succinic anhydride, phthalic anhydride), other activeesters such as N-hydroxysuccinimide esters, pentafluorophenyl esters anda variety of active esters in common use in amide bond formationincluding in the solid phase synthesis of peptide bonds. The —NH₂ groupsmay alternatively be capped by adding the corresponding free acids andan in-situ activating agent such as DCC, in common use in peptide-bondformation thereby creating an active ester in situ. Other reagents knownto be reactive towards —NH₂ groups can be used, such as alkylisothiocyanates (R—NCS), aryl isothiocyantes (Ar—NCS), alkylating agentsR-L (where L is a leaving group typically from the series Cl, Br, I,OS(O)₂R′ where R′ can be alkyl or aryl), Michael acceptors such asalpha-beta unsaturated carbonyl compounds (CHR═CH—CO— where R can be H,alkyl or aryl or substituted alkyl or aryl) or alpha-beta unsaturatedsulfones (CHR═CHS(O)₂R′ or Ar where R can be H, alkyl or aryl orsubstituted alkyl or aryl), sulfonating agents (such as RSO₂Cl) andderivatives thereof. In a similar manner, the —NH₂ groups can be cappedby reaction with active esters of carbonates of the general formulaRO—C(O) L, where L is described as above.

-   Reduction: In one preferred embodiment of the invention, the bond    linking the monosaccharide to the linker (for example compound G or    I of FIG. 1) is reduced using a reducing agent. However, in other    embodiments of the invention the methods do not comprise such a    reduction step. The bond may be reduced by a variety of well known    reducing agents, as known by one skilled in the art. If the bond    linking the monosaccharide to the linker is a C═N bond, preferably    the reducing agent is capable of saturating the double bond while    placing a hydrogen atom on the N.

Of special value are boranes or borohydrides comprising a BH bond,examples include NaBH₄, NaCNBH₃, and BH₃ complexes such as BH₃-pyridine,BH₃-dimethylsulfide or the like. Silanes with the structures R₃SiH canalso be used, such as silanes comprising SiH bonds, as can hydrogentransfer agents such as diimides, or homogeneous hydrogenation catalystsor hydrogenation catalysts comprising a metal-H bond.

The reduction results in a reactive monosaccharide sugar containing thestructure SugarCH—NH— preferably linked to a solid support via a linker.In general, if the monosaccharide sugar was an aldehyde, then reductionwill result in a compound of the structure SugarCH₂—NH—. If themonosaccharide sugar was a ketone, then the reduction will result in acompound of the structure SugarCH—NH—.

In one embodiment of the invention, the capping step is carried out andthe then reduction step is carried out. In another preferredimplementation of the method, the order of the capping and reductionsteps is reversed.

The reduction may in one embodiment be performed in situ, meaning that areducing agent (such as NaCNBH₃) may be added to the solid supportsimultaneously with the monosaccharide. It is thus comprised within thepresent invention that the sample comprising the monosaccharide may beincubated with the solid support and the reducing agent simultaneously.

(iv) Allowing the Detection Agent to Bind the Monosaccharide

Stage (iv) of the method of the present invention comprises the step ofallowing the detection agent to bind the monosaccharide.

This reaction of the TAG-R-Boronate with the captured monosaccharide isexemplified in FIG. 3:—The captured monosaccharide bonded to the solidsupport (any of G-L) is contacted with a solution of a functionalizedboronic acid of the general formula TAG-R-Boronate (M), where R ispreferably aromatic or heteroaromatic. The “TAG” can be any moiety thatpermits the specific detection of the Tagged species, eitherdirectly—such as by using a fluorescent TAG—or indirectly—for example bymeasuring a mass change attributed to a heavy metal TAG. Afterincubation with any of G-L, the TAG-R-Boronate (M) will stay covalentlyattached to the monosaccharide-solid support (such as, for example viaat least one OH-group) and optionally, the unbound TAG-R-Boronate can bewashed away. This leaves a solid material referred to asTAG-R-Boronate-adduct (N). In the absence of attched Glycose, theTAG-R-Boronate will not remain attached to the solid support and will bewashed away from the solid support that will then not have any attachedTAG.

Optionally, the method according to the present invention comprises afurther step after step (iv), wherein unbound detecting agent isremoved, preferably by washing. Thus, in one embodiment the taggedimmobilised terminal monosaccharide (e.g. compound N, FIG. 3) is washedprior to any further manipulations. Thus any amount of unbound TAG isremoved. Washing may easily be accomplished because the taggedmonosaccharide is immobilised on a solid support.

After washing, only covalent bound TAG will be present. Thus the amountof TAG will be correlatable to the amount of immobilised sugar.Accordingly, by determining the presence of TAG, the amount ofimmobilised sugar may be determined. If essentially all monosaccharidein a given sample was immobilised, the methods therefore in one aspectallow determining the amount of monosaccharide present in a sample.

The skilled person will readily be able to identify suitable washingconditions for a given tagged, monosacchairde (e.g. compound N, FIG. 3).The washing may for example be done with a solvent selected from thegroup consisting of water, aqueous buffer, organic solvents and mixedaqueous and organic solvents. The solvent may also be any of theaforementioned comprising one or more additives such as salts, divalentmetal cations, detergents, complexing agents includinginclusion-complex-forming molecules such as cyclodextrins orcalixarenes, chelating agents (for example EDTA), borates, boronates orsilicates. Furthermore, the solvent may optionally comprise detergentsand denaturing agents. The washing my be performed at any temperature,but preferably at temperatures in the range of 0-100° C.

(v) Detecting Detection Agent having Bound to the Monosaccharide

Stage (v) of the method of the present invention comprises the step ofdetecting detection agent having bound to the monosaccharide.

This detection step can allow conclusions to be drawn in relation to theidentity of the terminal monosaccharide cleaved from the glycosylatedsubstrate A, for example the detection step may allow allowidentification of at least one of the terminal monosaccharide(s) on theglycosylated substrate. One preferred identification method is viaindirect identification of the monosaccharide according to the specificdetachment method used, for example if one uses a specific glycosidasein step (i) of the method, one can then infer the identity of themonosaccharide detached based on the specificity of the glycosidaseused.

Many methods are known by one skilled in the art as suitable for use indetecting the detection agent. For example, the detecting step can becarried out by measuring absorbance or fluorescence. Thus, the detectingstep can be carried out by using spectrometry, such asfluorospectrometry.

It is preferred that the analysis allows quantification of at least oneof the monosaccharide(s) on the glycosylated substrate. Thus, the amountof TAG present on the TAG-R-Boronate adduct will indicate that amount ofmonosaccharide present, whose quantity can therefore be estimated usinga standard curve of captured reference monosaccharide. For example, thequantification may be carried out by comparing the results obtained withstandard curve obtained using different concentrations of a knownreference sample.

In one embodiment of the invention, the TAG molecule is released fromthe solid support before quantification.

This release step is exemplified in FIG. 4:—wherein the solubleTAG-R-Boronate (P) is released back into solution from theTAG-R-Boronate adduct (N) by contacting it with a solution of a cleavingagent comprising a competing ligand (O) The TAG-R-Boronate (P) releasedinto solution can then be estimated by solution methods that detect theTAG, such as spectroscopy. The TAG will no longer be detectable on theresidual solid-support which will have the structure according to any ofG-L.

The cleaving agent (O) can for example be a soluble compound thatcompetes with the immobilized monosaccharide and makes covalent bonds tothe boronate moiety. Such compounds include polyalcohols like glycerolor glucitol, or amino-alcohols like diethanolamine. Under certainconditions, water or alcohols may act as competing ligands.

In one embodiment of the invention a predetermined amount of a referencestandard is added to the sample comprising the glycosylated substrate.This may facilitate quantification of any released monosaccharide afterimmobilisation, and/or validate the efficiency of the capture-beads in acomplex sample. The reference standard can for example be a compoundcapable of reacting with —NH₂. Preferably the reference standard is analdehyde or a ketone, more preferably a sugar, such as a monosaccharide.

The present invention surprisingly discloses a high affinity betweenboranate and immobilised terminal monosaccharides. Accordingly, themethods of the invention are sufficiently sensitive in order forrelatively insensitive methods to be used for detection. Accordingly,surprisingly in one preferred embodiment of the invention the detectionmay be visual detection by eye. Visual detection by eye may for examplebe performed as described in Example 1 and disclosed in FIG. 13 herein.

Repeating the Method

The method described herein may advantageously be repeated at leastonce, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. This can forexample be advantageous should one wish to use the methods describedherein for monosaccharide sequencing of a glycosylated molecule, such asa polysaccharide. The repetitions of any of the methods disclosed hereinmay be carried out in parallel on different samples of glycosylatedsubstrates and/or using different methods for detaching themonosaccharide(s) from said glycosylated substrate(s). For example, onemay repeat the method using one or more different specific glycosidases(optionally on parallel samples), in order to indirectly determine theidentity of the relevant monosaccharides through the specificity of theglycosidase used.

Thus, the present invention provides in one aspect a method foroligosaccharide or polysaccharide sequencing, comprising subjecting apolysaccharide or molecule comprising a polysaccharide to any of themethods disclosed herein.

Method of Diagnosis of a Disease

The methods of the present invention may be used to identify particularglycosylation patterns associated with certain pathological conditions,thus in a further aspect of the present invention is disclosed a methodof diagnosis of a disease associated with abnormal glycoproteinglycosylation, comprising subjecting a sample of glycoproteins obtainedfrom a patient to any of the methods as disclosed herein. Identificationof one or more of the monosaccharides on the glycosylated substrate ofinterest can for example allow diagnosis of a specific glycoylationpattern associated with a particular disease class. Preferred diseasesassociated with an abnormal glycosylation patterns include, but are notrestricted to, Carbohydrate-Deficient Glycosylation Syndromes (CDGS),CDGS I, CDGS II, CDGS III, CDGS IV, neuronal diseases, diabetes,Tay-Sachs disease and cancers.

Method for Monitoring for Bacterial Contamination of Products

The method of the present invention may also be used advantageously toassay for bacterial contamination of products. Thus, in another aspectof the present invention is provided a method for monitoring forbacterial contamination of products, such as food, beverages orpharmaceutical products, comprising subjecting a sample of said productto one of the methods disclosed herein. This may be particularlyadvantageous for e.g. checking the quality of therapeutic glycosylatedproteins, as disclosed in e.g. Sinclair et al., “Glycoengineering: theEffect of Glycosylation on the Properties of Therapeutic proteins”.Thus, in one preferred embodiment of the present invention is provided amethod for characterising therapeutic glycosylated proteins, comprisingcarrying out any of the methods describe herein.

Covalent Adduct

When carrying out the method of the present invention, a covalent adductis formed between the monosaccharide and the detection agent, asdisclosed herein. Thus, in one aspect of the present invention isdisclosed a covalent adduct formed between a monosaccharide captured onthe solid phase, such as any of the monosaccharides disclosed herein,and a detection agent with formula X:

TAG-R-Boronate;

such as any of the detection agents disclosed herein.

Preferably, the covalent adduct is covalently bound to a capture groupon a solid support.

Fluorescent Compounds

When carrying out the method of the present invention, it is preferredthat fluorescent compounds are used as the TAG component of thedetection agent. Thus, in one aspect of the present invention isdisclosed novel fluorescent compounds useful as TAG compounds, which canbe used to produce the detection agents used in the present invention.Any of the fluorescent compounds disclosed herein may be used: twopreferred fluorescent compounds have the following structures:fluorescent hydroxymethyl-boronate 1 (shown in FIG. 11) and fluorescentnitro-boronate 2 (shown in FIG. 12)

Methods for manufacture of any of the fluorescent compounds disclosedherein are well-known to those skilled in the art, for example asdisclosed in e.g. “Boronic Acids: Preparation, Applications in OrganicSynthesis and Medicine”, edited by Dennis G. Hall, pub. Wiley-VCH VerlagGmbH & Co. KGaA, Weinheim, in particular chapter 1: “Structure,Properties, and Preparation of Boronic Acid Derivatives. Overview ofTheir Reactions and Applications”.

Kit of Parts

In another aspect of the present invention is disclosed a kit of partssuitable for using in the methods of the present invention, said kit ofparts comprising at least one solid support (such as any of the solidsupports disclosed herein), at least one capture group (such as any ofthe capture groups disclosed herein), and one or more detection agents,such as any of the detection agents disclosed herein. Said kit of partsmay also further comprise at least one specific glycosidase and/or asample of glycosylated substrate, preferably for use as a positivecontrol in quantitative or qualitative assays.

EXAMPLES

The following are illustrative examples of the methods of the inventionand should not be considered as limiting for the invention.

Abreviations used:

-   AMP-CPG: aminopropyl controlled pore glass-   DMF: dimethylformamide-   DIPEA: diisopropylethylamine-   TBTU: N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium    tetrafluoroborate-   DCM: dichloromethane-   DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene-   6-TRITC: 6-tetramethylrhodamine isothiocyante-   ES-MS: electrospray mass spectrum-   EDC: N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide-   BSA: bovine serum albumin-   rt: room temperature

Example 1 Synthesis of Capture Beads (FIG. 10)

Aminopropyl controlled pore glass (AMP-CPG, Millipore Prod. No. AMP1400B) (500 mg, loading of amino groups, ca 50 μmol/g) was washed withDMF (3×2 mL), 50% DIPEA in DMF (3×2 mL) and DMF (3×2 mL). The beads werethen treated with a mixture of the hydroxylamine linker 3 (45 mg, 100μmol), TBTU (25 mg, 75 μmol) and DIPEA (13 μL, 75 μmol) in DMF (2 mL)for 2 h at rt. The beads were washed with DMF (3×2 mL) and DCM (3×2 mL).Unreacted amino groups were capped by treating the beads with 50% Ac₂Oin pyridine (2 mL) for 15 min at rt. Silanol groups (on the glasssurface) were capped by treating the beads with 5%dichlorodimethylsilane in toluene (2 mL) for 30 min at rt, followed by10 min incubation in dry MeOH. The silanol capping was repeated. Thebeads were washed with MeOH, DMF and DCM. A small portion of the beadswas removed and the loading was determined to be ca 35 μmol/g based onthe absorbance of the Fmoc group released on treatment with 10% DBU inDMF, by comparison with a standard curve prepared using knownconcentrations of cleaved Fmoc group.

Synthesis of fluorescent hydroxymethyl-boronate 1 (FIG. 11)

6-TRITC (4, 7.4 mg, 17 μmol) was added to a solution of5-amino-2-hydroxymethyl phenyl boronic acid (5, Combi-Blocks Prod. No.BB-2043, 3.1 mg, 17 μmol) in 0.1 M NaHCO₃/Na₂CO₃ buffer pH 9 (1 mL) andDMF (1 mL). The mixture was stirred over night at rt in the dark. Themixture was concentrated under vacuum and the crude product wasdissolved in water, adsorbed on a Sep-Pak cartridge (C-18) which waswashed with H₂O. Elution with 30-50% MeCN in H₂O (containing 10% 0.1 MHCl) gave 1 as a purple solid (6.3 mg, 64%). ES-MS, m/z found 593.2([MH]⁺ calcd 593.2).

Preparation of ethylenediamine extended lissamine 7 (FIG. 12)

To a solution of ethylenediamine (232 μL, 3.47 mmol) in DCM (5 mL) wasadded Lissamine rhodamine B sulfonylchloride (6, mixture of 5/6 isomers,100 mg, 0.17 mmol) in one portion. The mixture was stirred over night atrt in the dark. The mixture was concentrated under vacuum and the crudeproduct was purified by adsorption on a Sep-Pak cartridge (C-18) whichwas washed with H₂O. Elution with 30% MeCN in H₂O (containing 10% 0.1 MHCl) gave 7 as a purple solid that was used without furthercharacterisation.

Synthesis of fluorescent nitro-boronate 2 (FIG. 12)

To a suspension of (3-carboxy-5-nitrophenyl)boronic acid (8,Combi-Blocks Prod. No. BB2476, 1.76 mg, 8.33 μmol), HOBt.H₂O (1.13 mg,8.33 μmol), EDC.HCl (1.60 mg, 8.33 μmol) and DIPEA (1.45 μL, 8.33 μmol)in dry DCM (1 mL) was added 7 (5.00 mg, 8.33 μmol). The mixture wasstirred for 2 h at rt in the dark. More DCM (5 mL) was added and themixture was washed with sat. NaHCO₃ and 0.1 M HCl. The organic phase wasconcentrated under vacuum and the crude product was purified by drycolumn vacuum chromatography using 50% MeOH in CHCl₃ (containing 1% 0.1M HCl). The fractions containing the product were concentrated andredissolved in DCM (5 mL) and washed with half sat. aq. NaHCO₃. Theorganic phase was dried (Na₂SO₄), filtered and concentrated givingproduct 2 as a purple solid (4.7 mg, 70%). ES-MS m/z found 816.3([MNa]⁺, calcd 816.2)

Preparation of bovine serum albumin (BSA) for enzyme digestion

BSA (5×2 mg/mL ampules, Pierce #23209) was dialyzed twice against water(5 L), and concentrated to 20 mg/mL in a centricon-30 ultrafiltrationtube.

Preparation of β-galactosidase for glycoprotein digestion

A centricon-30 ultrafiltration tube was preconditioned by washing withwater (2 mL), then spun for 8 min at rt to clean the membrane.Commercial β-galactosidase from bovine testes (Sigma, G4142) wasdissolved in buffer (0.1 M citrate/phosphate, pH 5.0) and 2 mL was addedto the Centricon tube. The tube was spun at 4000×g until 50 μL remained.The filtrate was discarded, and the retentate made up to 2 mL withbuffer. This was centrifuged again until 50 μL remained. The entireprocess of discarding the filtrate, adding buffer to 2 mL andcentrifuging was repeated 4 more times. After discarding the finalfiltrate, the tube was inverted and spun for 2 min at 1000×g, yielding asolution of the enzyme in buffer ready for use.

Digestion of Proteins and Glycoprotein Samples with Beta-Galactosidase

Solutions of 10 mg/mL of the proteins (fetuin (Sigma F3004),asialo-fetuin (Sigma A4781) and the dialyzed BSA described above, wereincubated in buffer at 37° C. overnight, both with and without addedbeta-galactosidase (0.23 U/mL). Six YM-10 ultrafiltration tubes wereprepared by washing the membranes with pure water (500 μL, centrifuging30 min at 14,000×g) to remove any glycerol from the membranes, removingthe retentate by shaking, and replacing the receiving tube with a freshreceiving tube. Then 200 μL of each of the six incubation solutions wasadded to a freshly prepared YM-10 tube which was centrifuged for 45 minat 14,000×g. The filtrates from each tube were used for the captureexperiments, and are referred to as Fet− (for the solution of Fetuinwithout added galactosidase), Fet+ (for the solution of Fetuin withadded galactosidase), AFet− (for the solution of asialo-fetuin withoutadded galactosidase), AFet+ (for the solution of asialo-fetuin withadded galactosidase), BSA− (for the solution of BSA without addedgalactosidase) and BSA+ (for the solution of BSA with addedgalactosidase).

Capture of Galactose on Capture-Beads.

All manipulations were performed under a stream of argon. Beads (10 mg,0.35 μmol of Fmoc-protected capture groups, FIG. 10) were placed in eachof eight glass test tubes and each was Fmoc-deprotected by treatmentwith 10% DBU in DMF (200 μL) for 20 min. The cleaving solution wasdecanted and the beads were washed with DMF (3×2 mL) and 0.1 Mcitrate/phosphate buffer pH 5 (3×1 mL) with decanting of each of thewashes.

To one tube each of the resulting hydroxylamine Capture Beads (FIG. 10)was added 50 μL of Fet−, Fet+, AFet−, AFet+, BSA−, BSA+, galactose (2mM) in buffer (referred to as Gal-std), and buffer alone (referred to asBlank). More buffer (50 μL) was added to each tube. The tubes werestoppered and incubated overnight at 60° C. in a heating block. Aftercooling to rt, remaining capture groups were capped by adding 50% aceticanhydride in MeOH and shaking gently for 20 min.

The remaining manipulations were no longer performed under argon. Thebeads from each tube were transferred to a plastic syringe equipped witha Teflon frit, and washed by aspiration with buffer (3×0.5 mL), H₂O(3×0.5 mL), MeOH (3×0.5 mL), H₂O (3×0.5 mL), 5% DIPEA in DMF (0.5 mL),DMF (3×0.5 mL), H₂O (3×0.5 mL) and MeOH (3×0.5 mL). A portion of eachbatch of beads was dried under vacuum.

Staining and visual detection of captured galactose on treated CaptureBeads after treatment with fluorescent hydroxymethyl-boronate 1 (FIG.13)

The dried capture beads (2 mg) described above that had been exposed toeach of Fet−, Fet+, AFet−, AFet+, BSA−, BSA+, Gal-std and Blanksolutions, were washed with DMF (1×0.5 mL) and treated with a mixture of0.5 mM 1 (FIG. 11) in DMF (100 μL) and 0.1 M carbonate buffer pH 9 (100μL). The resulting samples were shaken gently for 1 h at rt. The beadswere washed with DMF (3×0.5 mL), 0.1 M carbonate buffer pH 9 (1×0.5 mL),H₂O (1×0.5 mL), DMF (3×0.5 mL), DCM (3×0.5 mL). Portions of the beadswere transferred to small glass vials and photographed with a hand-helddigital camera (FIG. 13, top panel).

Release of fluorescent boronate from stained beads (FIG. 13)

A solution of glycerol/MeOH/H₂O (1:2:2, v/v/v, 100 μL) was added to theeach of the tubes containing beads shown in the top panel of FIG. 13.The samples were shaken gently for 1 h. The supernatants were removedfrom each tube and transferred to fresh plastic Eppendorf tubes. Thesupernatants from the tubes designated AFet+ and Gal-std were bright redwhile the supernatants from the other beads were clear (FIG. 13, bottompanel). Washing of the residual beads once more with glycerol/MeOH/H₂O(1:2:2, v/v/v, 100 μL) left the beads designated AFet+ and Gal-std whiteagain, indistinguishable from the others (FIG. 13, middle panel).

(These Examples exemplify use of compound 1 shown in FIG. 11; Compound 2shown in FIG. 12 was found to be equally effective to Compound 1 for thestaining and detection of galactose captured on Capture Beads, and wasequally efficiently released on treatment with glycerol.)

1-63. (canceled)
 64. Method for analysis of a terminal monosaccharide ona glycosylated substrate, said method comprising the steps of: (i)detaching said monosaccharide from said glycosylated substrate using anexoglucosidase; (ii) allowing said detached monosaccharide to covalentlybind to a capture group on a solid support; (iii) incubating saidcovalently bound monosaccharide with a detection agent with formula X:TAG-R-Boronate wherein TAG=a tag moiety capable of being detectedR=organic moiety Boronate=a boronic acid moiety or ester thereof, saidboronate being attached to a carbon atom comprised in said R group; (iv)allowing the detection agent to bind the monosaccharide (v) detecting,directly or indirectly, detection agent having bound to themonosaccharide.
 65. The method according to claim 64 comprising afurther step after step (iv), wherein unbound detecting agent isremoved, preferably by washing.
 66. The method according to claim 64,wherein said capture group is attached to the solid support via a linkergroup.
 67. The method according to claim 64, wherein a capping agent isbound to unbound capture groups before step (iv)
 68. The methodaccording to claim 64, wherein the glycosylated substrate to be analysedis comprised within a sample, and wherein said method comprises anadditional step after step (i), wherein components of said sample areremoved, such as by size exclusion, preferably by ultrafiltration or bydialysis.
 69. Method for polysaccharide sequencing, comprisingsubjecting a polysaccharide or molecule comprising a polysaccharide tothe method according to claim
 64. 70. Method of diagnosis of a diseaseassociated with abnormal glycoprotein or glycolipid glycosylation,comprising subjecting a sample of glycoproteins or glycolipids obtainedfrom a patient to the method according to claim
 64. 71. Method formonitoring for bacterial contamination of products, such aspharmaceutical products, comprising subjecting a sample of said productto the method according to claim
 64. 72. Covalent adduct formed betweena monosaccharide and a detection agent with formula X: TAG-R-Boronatewherein TAG=a tag moiety capable of being detected R=organic moietyBoronate=a boronic acid moiety or ester thereof, said boronate beingattached to a carbon atom comprised in said R group.
 73. The covalentadduct according to claim 64, wherein said detection agent is selectedfrom those described in FIGS. 5, 6, 7 or
 8. 74. Fluorescent compoundwith formula according to any of the fluorescent compounds in FIG. 11 or12.
 75. Kit of parts comprising at least one solid support, at least onecapture group, and a detection agent.
 76. The kit of parts according toclaim 75, wherein said detection agent has a formula selected fromTAG-R—B(OH)₂, TAG-R—B(OH)(OR′) or TAG-R—B(OR′)(OR″) wherein R′ and R″may be either aliphatic or aromatic and are optionally covalentlyattached to R or from any of the groupings disclosed in FIGS. 5, 6, 7,8, formula 1 or 2 shown in FIGS. 11 and 12 or comprises aryl boronate orheteroarylboronate.
 77. The kit of parts according to claim 75, furthercomprising at least one specific glycosidase.
 78. The kit of partsaccording to claim 75, further comprising a sample of glycosylatedsubstrate.